Effect Of Short TRPM8Variant Alpha On Biological Behavior And The Mechanism Of Regulation In Prostate Cancer LNCaP Cells In Vitro

Chapter1. The reconstruction,amplification and sequencing and expressive identification of sM8a eukaryotic expression plasmid in vitroObjective To detect the expression of sM8α in prostate cancer LNCaP, PC-3, DU145cells, and reconstruct the eukaryotic expression plasmid (pcDNA3.1(-)-sM8a-His) and sequence. Then transfect pcDNA3.1(-)-sM8a-His reconstructive plasmid into androgen dependent prostate cancer LNCaP cells and western bloting for the expression of sM8α-His fusion protein. It provides the basis of investigation of overexpression of sM8a in LNCaP cells.Methods1) Reverse transcription polymerase chain reaction (RT-PCR) is performed for detection of the expression of sM8a in prostate cancer LNCaP, PC-3, DU145cells. High fidelity PCR is chosen for sM8α-His fragment, which is digested by two kinds of restriction enzymes (EcoR Ⅰ and Xho Ⅰ) and is linked by T4DNA ligase for pcDNA3.1(-)-sM8a-His reconstructive plasmid, and gene sequences will be blasted on pubmed after sequencing.2)incubate293T cells for transfection of pcDNA3.1(-)-sM8a-His plasmid and control plasmid transfection into293T cells. After48hours, the expression of sM8α-His fusion protein is identfied by western-bloting.Results We detected sM8a mRNA expression with low level in prostate cancer LNCaP, PC-3,DU145cells. The pcDNA3.1(-)-sM8a-His plasmid was successfully reconstructed and was sequenced completely correct. After transient transfection of pcDNA3.1(-)-sM8a-His plasmid into293T cells, we detected the expression of sM8α-His fusion protein.Conclusion The sM8a-His fusion protein can be expressed by plasmid of pcDNA3.1(-)-sM8a-His in eukaryotic293T cells, and it provides the preliminary basis of effect of sM8a in prostate cancer LNCaP cells. Chapter2. Effect of short TRPM8variant alpha (sM8a) on biological behavior of prostate cancer LNCaP cells in vitroObjective To detect the effect of overexpression sM8α on proliferation, cell cycle, apoptosis, migration, invasion and distribution in LNCaP cells after transfection and expression of pcDNA3.1(-)-sM8α-His plasmid and empty vector.Methods Androgen dependent prostate cancer LNCaP cells is incubated in six-wells plate. According to the G418concentration of300ug/ml,400ug/ml,500ug/ml,600ug/ml,700ug/ml,800ug/ml, optimal screening concentration of G418is found for stable transfection. Cells is incubated and divided into three groups:normal group, control group and experimental group. The interventions are respectively without plasmid, with empty controlled plasmid, with pcDNA3.1(-)-sM8a-His plasmid. Using lipofectamine2000for transfection, then cells are screened in optimal concentration of G418. Stable transfection cells are harvested by identification of RT-PCR and western-bloting. The method of MTT is used for proliferation of cells from the first day to fifth day, then cell growth curve is drawn. Apoptosis induced by starvation for24hours and cell cycle are detected by flow cytometry. Migration and invasion of LNCaP are measured by transwell. The expression of sM8α-His fusion protein is intracellular located by quantum dots-based immunofluorescent imaging.Results The optimal screening concentration of G418is700ug/ml. After successful indentification of stable transfection cells, overexpression of sM8a can improve the potential of migration an invasion of prostate cancer LNCaP cells compared with control group. The apoptosis rate induced by starvation is decreased in experimental group. There are no differences in cell proliferation and cell cycle and the location of overexpression of sM8a-His fusion protein is in cytoplasm.Conclusion sM8a can regulate the migration and invasion of LNCaP cells and may play an important role in anti-apoptosis. The intracellular location of overexpression of sM8a-His fusion prostein is in cytoplasm. Chapter3. The realationship between sM8a and mitogen-activated protein kinases (MAPK) signaling pathway in prostate cancer LNCaP cellsObjective To investigate the role of MAPK signal pathway, including p38, JNK, ERK and phosphorylation protein, MMP2, MMP9, full size TRPM8in prostate cancer LNCaP cells after overexpression of sM8a,Methods Normal LNCaP cells, LNCaP-NC, LNCaP-sM8a cells are cultured and collected, then RIPA and phosphorylase inhibitor are added for lysing the cells. Proteins are extracted by sonication and centrifuge for western-bloting.Results The positive outcomes are downregulation of p-JNK protein and activation of MMP2in LNCaP-sM8a cells.The expression of total p38, JNK, ERK and p-p38, p-ERK protein, MMP9and full size TRPM8, have no differences among normal LNCaP cells, LNCaP-NC and LNCaP-sM8a cells.Conclusion JNK/MAPK signaling pathway plays an important role in LNCaP cells of sM8a overexpression and the activation of MMP2may improve the probability of migration and invasion of LNCaP cells of sM8a overexpression.